Glycoconjugate vaccines date their Industrial development for human use back to 1990, on the basis of theoretical studies and molecular models originally reported between 1929 and 1940. Those studies were re-evaluated and advanced at molecular level in the decade 1980-1990, taking advantage of the newer methodologies under development in the biotechnology Era.
The strategy of using as a vaccine a semi-synthetic antigen involving a carbohydrate covalently bound to a carrier protein, have had the experimental base in the demonstration that the immune system of human infants is not completely mature until over 2 years of age, so that highly purified Polysaccharide antigens are not efficiently recognized as foreign antigens by the human host. As a result, an adequate amount of functional, anamnestic, IgG antibody do not follow to the injection of multiple Polysaccharide injections. The carrier protein does have the function to properly prime and boost the population of helper T-cells of the host's immune system which then results in the expansion of the B-memory cell secreting serum IgG antibodies specific for the carried Polysaccharide as well as for the carrier Protein. The impact of glycoconjugate vaccines on Public Health since the early Nineties, has been invaluable as they have saved millions of children's lives at risk of deadly acute infections, like spinal meningitis due to Gram-positive and Gram-negative bacteria (Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis).
Several strategies of chemical conjugation are known for industrially preparing a protein-carbohydrate conjugate antigen. The known techniques are unsatisfactory as to the quantitative yields.
In accordance to an aspect, the present Application deals with a preparation method which provides for:    A) the use of rigorously Endotoxin-free (LPS-free) Polysaccharide antigens, to be conjugated to a carrier protein, for an improved purity of the antigens as well as for their improved safety behaviour;    B) a quantitative yield of each reaction step involved in the process, which then results in an overall yield of the glycoconjugate antigen quantitative with respect to both components of the conjugate, that is the carrier protein and the carried carbohydrate;    C) a flexible conjugation procedure. Specifically, the conjugation process allows the synthesis of glycoconjugates as mono-, bi- or poly-valent antigens able to simultaneously express mono-, bi- or poly-valent immunogenic characteristics.
In accordance to an aspect, the present invention provides for purification and or conjugation methods as hereinafter described and claimed in the appended claims. In particular, an embodiment of the present invention provides for a conjugation process which involves the following steps:    A. Activation of the endotoxin-free polysaccharide antigen to polyfunctional polysaccharide through a diamino-alkyl spacer introduced via:            A1. O-de-hydrogen uncoupling obtained by introduction of reactive carbonyl groups with an oxidative agent to generate aldehyde groups in the presence of borate ions when the reaction is performed in aqueous solvent; such groups being then reacted with the diamino-alkyl spacer in the presence of a reducing agent,        A2. linkage of the diamino-alkyl spacer to the already present reactive carbonyl residues in the form of carboxyl groups by water-insoluble carbodiimide, in the presence of organic solvents,            B. Activation of the immunogenic carrier protein via bis-succinimidyl ester of an aliphatic bicarboxylic acid, resulting in a poly-functional protein through monosuccinimdylesters of the Lysine residues,            C1. Coupling of the activated poly-functional carrier protein to the activated endotoxin-free poly-functional polysaccharide, via the monosuccinimidylesters introduced on the Lysine residues of the protein and the amino groups introduced on the polysaccharide; or, alternatively,        C2. Coupling of the poly-functional amino-activated polysaccharide to the carrier protein via a bis-succinimidyl ester of an aliphatic bicarboxylic acid which reacts sequencially, in the same reaction mixture, with the amino groups of the amino-activated polysaccharide and with the epsilon-amino groups of the Lysine residues of the protein.        
Preferably the polysaccharide antigens are purified by a method which involves the removal of contaminating endotoxin by affinity-binding of the lipid A moiety of LPS with synthetic anti-endotoxin peptides (SAEP) having retro-inverted amino acid sequences, which results in polysaccharides having a content of endotoxin conveniently lower than 0.125 Endotoxin Unit/μg polysaccharide, equivalent to less than 0.00125% (w/w).